The background information provided here is meant solely to assist the reader in understanding the current invention and the advances in the art provided thereby. Nothing in this section is intended, nor is it to be construed as, prior art to this invention.
In any genome, i.e., the accumulation of DNA that encodes the biological information in the form of genes that, along with environmental and developmental factors, determine the phenotype of all living things, there may be several kinds of genetically important allelic variations classified as insertions, deletions, single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). These variations may be harmless or they may give rise to serious potentially lethal disorders, which includes well-known diseases such as cystic fibrosis, sickle cell anemia, Tay-Sach's disease, hemophilia, Crohn's disease, heptatitis C, AIDS and cervical cancer.
With regard to disease-causing organisms, genotypic variation can result in either more or less serious infection and/or greater or lesser susceptibility to treatment, in general or with regard to specific treatment regimes. A prime but non-limiting example of this is Human Papillomavirus (HPV) for which over 200 genotypes are known. Of these, most relate to relatively benign problems, in particular warts. On the other hand a handful of HPV genotypes have been associated with cancer, particularly cervical cancer. These cancer-related genotypes have been subdivided into two groups, high-risk and low risk. The high-risk genotypes include HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. The low-risk HPV genotypes include HPV-6, 11, 34, 40, 42, 43 and 44. It is not uncommon for an individual to be infected by several of these genotypes simultaneously. Each of these genotypes may, however, react very differently to specific treatment regimes. For example, the much-touted vaccine against cervical cancer-causing HPV is only effective against HPV 6, 11, 16 and 18, that is, two low-risk and two high-risk genotypes.
Another prime example of multiple variant pathogens is hepatitis C(HCV). HCV consists of 22 variants, 1a, b, c; 2a, b, c; 3a, b; 4a, b, c, d, e; 5a; 6a; 7a, b; 8a, b; 9a, 10a and 11a. These are all similar enough to be considered HCV but they are distinct genotypes.
The primary method of determining individual genotype(s) among a set of possibilities is to sequence DNA isolated from the source, i.e., from the viruses infecting a patient in the case of HPV.
DNA sequencing is typically accomplished using one of a number of procedures based on primer hybridization at or near a region of interest on a DNA, usually single-stranded. The hybridized primer is then extended over the region of interest followed by analysis of the resulting fragments to obtain the nucleotide sequence of the region of interest and, it is hoped, from that to identify the DNA and the source from which it was obtained.
Numerous sequencing techniques are known although most of them are based fundamentally on the Sanger dideoxy procedure, which harkens from 1975. The Sanger dideoxy procedure, however, requires the isolation of a sample containing a single DNA. Any more than that and the resulting data obtained are uninterpretable.
Sequencing by hybridization is also possible. In this technique, a pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array of known sequences. A strong hybridization signal from a particular location on the array identifies the unknown sequence as being present in the subject DNA.
It would be extremely beneficial to have a means of rapidly identifying one variant from among a possible plurality of variants or even more beneficial or essentially simultaneously and unambiguously identify more than one of the variants present. With regard to pathogenic organisms, this would permit rapid identification of variant-specific treatment regimes. The present invention provides such a means.